Liquid-containing substance analyzing device and liquid-containing substance analyzing method

ABSTRACT

A liquid-contained substance analysis device according to this invention comprises: a substrate  14,  which is coated on the surface with PVA and which is mirror finished; a first light source  18  for illuminating light onto substrate  14;  and light detector  30,  which detects the scattered light due to a liquid or a trace of a liquid that exists on substrate  14,  and the position of liquid L or trace of liquid L is detected based on the scattered light detected by light detector  30.  By then illuminating excitation light from a second light source  20  onto the detected position, the substances contained in liquid L are analyzed.

TECHNICAL FIELD

[0001] This invention concerns a liquid-contained substance analysisdevice and a liquid-contained substance analysis method for analyzingsubstances contained in a liquid, and particularly concerns those usedfor analysis of microvolumes of liquid.

BACKGROUND ART

[0002] Arts for analyzing substances contained in a liquid have existedsince priorly. As methods for specifying the position of a liquid thatis analyzed in such analysis of liquid-contained substances, methods ofusing microtiter plates and methods, in which markers for positioningare prepared in advance on a substrate onto which a liquid is to bedropped, have been known.

DISCLOSURE OF THE INVENTION

[0003] However, the abovementioned methods of specifying the position ofa liquid had the following problems due to degradation of the positionalprecision of the dropping of liquid as the quantity of liquid to beanalyzed became minute. That is, present microtiter plates are notsuitable for realizing high densities and, to begin with, cannot beapplied to microvolumes of liquid. With methods in which markers forpositioning are prepared in advance on a substrate onto which a liquidis to be dropped, since accurate dropping of a microvolume of the liquidonto the position at which a marker has been made was difficult, it wasdifficult to ascertain onto which position on the substrate the liquidhad been dropped.

[0004] Furthermore when the quantity of liquid is extremely minute, theliquid evaporates readily, and with types of liquid that evaporateimmediately upon being dropped onto a substrate, the position of liquidwas even more difficult to ascertain.

[0005] An object of this invention is thus to resolve the above problemsand to provide a liquid-contained substance analysis device and aliquid-contained substance analysis method, which enable the positionsof microvolumes of liquid to be ascertained smoothly and thereby enablesubstances contained in the liquid to be analyzed readily.

[0006] A liquid-contained substance analysis device according to thisinvention comprises: a mirror-finished substrate, onto which a liquidthat is to be analyzed is dropped; a first light source for illuminatingthe substrate; a light detector, detecting scattered light resultingfrom the scattering of light from the first light source by the liquidor trace of the liquid that has been dropped onto the substrate; aposition detector, detecting the position onto which the liquid has beendropped based on the scattered light detected by the light detector; asecond light source, illuminating the position detected by the positiondetector with light of a shorter wavelength than the light output fromthe first light source; a fluorescence detector, detecting fluorescenceresulting from the excitation of the liquid or trace of the liquid bythe light that has been illuminated from the second light source; and ananalyzer, analyzing substances contained in the liquid by thefluorescence detected by the fluorescence detector.

[0007] By thus illuminating light onto the mirror-finished substrate andusing the resulting scattered light from the liquid or trace of theliquid that exists on the substrate, the position at which the liquidexists can be detected readily. That is, since the mirror-finishedsubstrate will not give rise to scattered light, it can be judged thatthe liquid or trace of the liquid exists when scattered light occurs. Bythen illuminating light from the second light source onto the positionat which the liquid or trace of the liquid has been detected andanalyzing the fluorescence emitted by excitation of the substancescontained in the liquid, the substances contained in a microvolume ofliquid can be analyzed smoothly.

[0008] With the above-described liquid-contained substance analysisdevice, the second light source may be positioned so that the outputlight from the second light source intersects the normal to thesubstrate plane and the fluorescence detector may be positioned in thedirection of the normal to the substrate plane.

[0009] By thus making the light output from the second light source beincident so as to intersect the direction of the normal to the substrateplane and detecting the fluorescence excited by this light by means of afluorescence detector positioned in the direction of the normal to thesubstrate plane, the reflected light output from the second light sourcewill be prevented from becoming incident directly onto the fluorescencedetector, thereby enabling a weak fluorescence to be detected.

[0010] Also with the above-described liquid-contained substance analysisdevice, the surface of the substrate may be coated with a water-solubleorganic thin film.

[0011] When the substrate is coated with a water-soluble organic thinfilm, the substrate surface becomes modified at apart at which a liquidhas been dropped, thereby enabling the trace of the liquid to bedetected even when the liquid evaporates.

[0012] Also with the above-described liquid-contained substance analysisdevice, the substrate may be a silicon substrate on which a naturaloxidation film has been formed.

[0013] When such a silicon substrate, on which a natural oxidation filmhas been formed, is used, the substrate surface of a portion on which aliquid has been dropped becomes modified by the formation of a newsilicon oxide film, thereby enabling the trace of the liquid to bedetected even when the liquid evaporates.

[0014] The above-described liquid-contained substance analysis devicemay furthermore be equipped with a stage for setting the substratethereon and moving the substrate so that light from the first lightsource will be illuminated onto the substrate.

[0015] By thus placing the substrate on the stage and making thesubstrate movable, the illumination positions of the light output fromthe first light source and second light source that illuminate thesubstrate can be shifted.

[0016] A liquid-contained substance analysis method according to thisinvention comprises: a liquid dropping step of dropping a liquid to beanalyzed onto a mirror-finished substrate; a light illumination step ofilluminating light onto the substrate by means of a first light source;a scattered light detection step of detecting scattered light resultingfrom the scattering of light from the first light source by the liquidor trace of the liquid that has been dropped onto the substrate in theliquid dropping step; a position detection step of detecting theposition onto which the liquid has been dropped based on the scatteredlight detected in the scattered light detection step; a second lightillumination step of illuminating the position detected in the positiondetection step with light from a second light source that outputs lightof a shorter wavelength than the light output from the first lightsource; a fluorescence detection step of detecting the fluorescenceresulting from the excitation of the liquid or trace of the liquid bythe light illuminated in the second light illumination step; and ananalysis step of analyzing substances contained in the liquid based onthe fluorescence detected in the fluorescence detection step.

[0017] By thus illuminating light onto the mirror-finished substrate andusing the resulting scattered light from the liquid or trace of theliquid that exists on the substrate, the position at which the liquidexists can be detected readily. That is, since the mirror-finishedsubstrate will not give rise to scattered light, it can be judged thatthe liquid or trace of the liquid exists when scattered light occurs.

[0018] By then illuminating light from the second light source onto theposition at which the liquid or trace of the liquid has been detectedand analyzing the fluorescence emitted by excitation of the substancescontained in the liquid, the substances contained in the microvolume ofliquid can be analyzed smoothly.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 is a diagram, showing a liquid-contained substance analysisdevice of a first embodiment.

[0020]FIG. 2 is a flowchart, showing the operations of theliquid-contained substance analysis device of the first embodiment.

[0021]FIGS. 3A, 3B, 3C, and 3D are diagrams, showing images of thescattered light and fluorescence in the liquid-contained substanceanalysis device of the first embodiment.

[0022]FIG. 4 is a diagram, showing a liquid-contained substance analysisdevice of a second embodiment.

[0023]FIGS. 5A and 5B are diagrams, showing images of the scatteredlight and fluorescence in a case where a silicon substrate is used.

[0024]FIGS. 6A, 6B, 6C, and 6D are diagrams, showing images of thescattered light and fluorescence in a case where fluorescence analysisis performed in a batch.

BEST MODES FOR CARRYING OUT THE INVENTION

[0025] Preferred embodiments of liquid-contained substance analysisdevices according to this invention shall now be described in detailalong with the drawings. With regard to the description of the drawings,the same elements shall be provided with the same symbols and redundantexplanations shall be omitted.

[0026]FIG. 1 is a diagram, showing the arrangement of a liquid-containedsubstance analysis device (referred to hereinafter as “analysis device”)10. Analysis device 10 comprises a motor-driven movable stage 12, asubstrate 14, set on motor-driven movable stage 12, a liquid dropper 16for dropping a liquid L to be analyzed onto substrate 14, and a firstlight source 18 and a second light source 20, each of which illuminateslight onto substrate 14 and the liquid L that has been dropped ontosubstrate 14.

[0027] Substrate 14, which is set on motor-driven movable stage 12, ismirror finished. Light that is perpendicularly incident on substrate 14is thus reflected in the direction perpendicular to substrate 14. Thesurface of substrate 14 is also coated with a water-soluble thin film 14x.

[0028] Thin film 14 x is formed of a water-soluble organic thin film, inparticular, water-soluble PVA (polyvinyl alcohol).

[0029] First light source 18 is positioned to output light substantiallyparallel to the substrate plane. A first half-mirror 22, is positionedalong the output optical axis of first light source 18 and is tilted by45° with respect to the optical axis. The output light from first lightsource 18 is thereby bent at a right angle in the direction of substrate14. The output light bent by first half-mirror 22 is illuminated ontosubstrate 14. An objective lens 26 is disposed between first half-mirror22 and substrate 14, and the output light from first light source 18 isilluminated onto a predetermined region (hereinafter, this predeterminedregion shall be referred to as “measurement spot”) R on substrate 14 bymeans of objective lens 26.

[0030] Objective lens 26 is preferably a dark-field lens. This isbecause with a dark-field objective lens 26, the illuminated light willbe illuminated onto substrate 14 at an angle with respect to thesubstrate normal and the reflected light (from the substrate) that isdirected in the substrate normal direction will thereby be reduced toenable a scattered light image of high contrast to be obtained. A focaldepth adjustment optical device 28 is disposed along the output opticalaxis at a position near first light source 18 to provide an arrangementthat enables the first and second light of different wavelengths to beadjusted to be equal in focal depth.

[0031] Second light source 20 is a light source that outputs light of ashorter wavelength than first light source 18 and is an excitation lightsource for generation of fluorescence from liquid L. Second light source20 outputs light substantially perpendicular to substrate 14 and isdisposed so that its optical axis intersects the optical axis of theoutput light from first light source 18. At the position at which theoutput light from second light source 20 and the output light from firstlight source 18 intersect, a second half-mirror 24 is disposed in amanner that is tilted by 45° with respect to the output optical axis ofsecond light source 20, and the output light from second light source 20is thereby bent and made to progress along the same optical axis as theoutput light from first light source 18 and reach measurement spot R onsubstrate 14.

[0032] Motor-driven movable stage 12 can move substrate 14 so thatsubstantially the entire region of substrate 14 can be scanned by meansof measurement spot R. Also, liquid dropper 16, for dropping liquid L,which is the subject of measurement, is disposed above motor-drivenmovable stage 12, and motor-driven movable stage 12 can move substrate14 to a position at which liquid L is to be dropped by liquid dropper16.

[0033] Analysis device 10 comprises a light detector 30, which detectsscattered light resulting from the scattering, by the liquid L or traceof the liquid L that exists on substrate 14, of light output from firstlight source 18 and illuminated onto substrate 14, and a fluorescencedetector 32, which detects the fluorescence emitted from the liquid L asa result of the light output from second light source 20 and illuminatedonto the liquid L that has been dropped onto substrate 14.

[0034] Light detector 30 detects the scattered light scattered by theliquid L or a trace of the liquid L that exists on substrate 14. Lightdetector 30 is disposed above substrate 14 so as to lie along the samestraight line as substrate 14 and objective lens 26. An optical filter36, for cutting light from the exterior, is disposed in front of thelight detection surface of light detector 30. Between first half-mirror22 and optical filter 36, a half-mirror 34 is disposed in a manner thatis tilted by 45° with respect to the normal to the substrate plane andsplits the light from substrate 14 into a direction perpendicular to theoptical axis.

[0035] Fluorescence detector 32 is disposed along the optical axis ofthe split light. Fluorescence detector 32 detects the fluorescenceemitted by excitation of substances contained in liquid L as a result oflight output from second light source 20. Also, an optical filter 38,for cutting the reflected light of the excitation light output fromsecond light source 20, is disposed in front of the light detectionsurface of fluorescence detector 32.

[0036] Analysis device 10 furthermore has a computer 40. Computer 40 isconnected to first light source 18, second light source 20, motor-drivenmovable stage 12, and liquid dropper 16 and controls the respectiveoperations of these components. Computer 40 is also connected to lightdetector 30 and fluorescence detector 32, has the function of performingimage processing and data analysis based on the light detected by therespective detectors 30 and 32, and thus functions as a positiondetector and as an analyzer.

[0037] The operations of analysis device 10 shall now be described, andin accompaniment, a liquid-contained substance analysis method of thisembodiment shall be described. In the description that follows, therange onto which liquid L to be analyzed is dropped, that is the rangein which there is a possibility that liquid L or a trace of liquid Lexists on substrate 14 shall be referred to as the “measurement range.”

[0038] First, substrate 14, which has been mirror finished, is coatedwith a thin film 14 x (S10) and set on the upper surface of motor-drivenmovable stage 12. Substrate 14 is then moved by motor-driven movablestage 12 so that the measurement range will be set at the position ontowhich liquid L will be dropped by liquid dropper 16. Subsequently, amicrovolume of liquid L is dropped onto substrate 14 by means of liquiddropper 16 (S12).

[0039] Substrate 14 is then moved by motor-driven movable stage 12 sothat measurement spot R will be contained within the measurement range(S14). Motor-driven measurement stage 12 is stopped once, light fromfirst light source 18 is output, and light is illuminated ontomeasurement spot R of substrate 14 (S16). The scattered light that isscattered by the liquid L or the trace of the liquid L that exists onsubstrate 14 is then detected by light detector 30 (S18).

[0040] Based on the result of detection by light detector 30, it isjudged whether or not liquid L or the trace of liquid L exists withinmeasurement spot R of substrate 14 (S20). This judgment is made based onwhether or not scattered light has been detected. That is, sincesubstrate 14 is mirror-finished, the illumination light from first lightsource 18 will not be scattered by substrate 14.

[0041] Thus if scattered light is detected, it is judged that eitherliquid L or a trace of liquid L exists on substrate 14. If liquid Lexists on substrate 14, light illuminated onto substrate 14 is scatteredby this liquid L since liquid L acts as a mirror. Even if liquid L hasevaporated, since thin film 14 x, which had been coated onto the surfaceof substrate 14 dissolves and the surface of substrate 14 is therebymodified when liquid L is dropped, a trace of liquid L will remain andthe light illuminated by substrate 14 will thereby be scattered.

[0042] If as a result of judgment, it is judged that liquid L or a traceof liquid L exists, the scattered light is analyzed by computer 40 todetect the position of liquid L (S22) That is, if liquid L exists, sincethe illuminated light will be scattered by liquid L itself, the positionof liquid L can thereby be detected. Also, even if liquid L hasevaporated, since the surface of substrate 14 that has been modified bythe dropped liquid L bulges so as to surround the location at whichliquid L had existed, the position onto which liquid L had been droppedcan be detected by analysis of the scattered light.

[0043]FIGS. 3A and 3B illustrate specific examples. FIG. 3A is adiagram, showing an image of scattered lights detected by light detector30. By analyzing this scattered light S by means of computer 40, anoutline (liquid L trace) B on the surface of substrate 14 that has beenmodified by the dropped liquid L can be determined as shown in FIG. 3B.In this case, the position D onto which liquid L has been dropped is atthe inner side of outline B.

[0044] On the other hand, if it has been judged that neither liquid Lnor a trace of liquid L exists, the step (S14) of moving substrate 14 bymeans of motor-driven movable stage 12 is returned to. Here, in order sothat the entire measurement range of substrate 14 may be covered bymeans of measurement spot R, substrate 14 is moved so that themeasurement spot R after movement of substrate 14 will be adjacent tothe measurement spot R onto which light was illuminated previously.

[0045] After the position of liquid L or of a trace of liquid L has beendetected, light from second light source 20 is output and illuminatedonto the detected position D (S24). As shown in FIG. 3B, detectedposition D lies at the inner side of outline B. The fluorescence thathas been excited by liquid L or trace of liquid L is then detected byfluorescence detector 32 (S26). An image of the detected fluorescence Fis shown in FIG. 3C.

[0046] Since the fluorescence F that arises as a result of excitation ofsubstances contained in liquid L will exist at the position at whichliquid L or trace of liquid L has been detected as shown in 3D, in acase where the substances contained liquid L are to be analyzed,excitation light is illuminated onto the range of the detected positionD and studied. The fluorescence that has been detected by fluorescencedetector 32 is then analyzed and the analysis results are preserved bymeans of computer 40 (S28).

[0047] Subsequently, analysis device 10 judges whether or notmeasurements have been made for the entire measurement range (S30), andif measurements have been made for the entire measurement range, themeasurements are ended. If measurements have not been made for theentire measurement range, the step of moving substrate 14 by means ofmotor-driven movable stage 12 is returned to again (S14).

[0048] Since analysis device 10 of this embodiment has a first lightsource 18, which illuminates light onto a mirror-finished substrate 14,and a light detector 30, which detects the scattered light scattered byliquid L or a trace of liquid L that exists on substrate 14, if liquid Lexists on substrate 14 even by a minute amount, the position of liquid Lor trace of liquid L can be specified based on the detected scatteredlight. Analysis of an extremely minute amount of liquid L can thus becarried out smoothly.

[0049] Also with analysis device 10 of this embodiment, since a thinfilm 14 x is coated onto the surface of substrate 14 and when liquid Lis dropped, thin film 14 x dissolves and the surface of substrate 14 isthereby modified, even if liquid L evaporates, the light illuminated byfirst light source 18 will be scattered and the position on to whichliquid L has been dropped can be detected by means of this scatteredlight.

[0050] Also with analysis device 10 of this embodiment, since firstlight source 18, which illuminates light for detection of the positionof liquid L, uses light that is longer in wavelength than the light ofsecond light source 20, the liquid L that is subject to analysis willnot be destroyed.

[0051] With the liquid-contained substance analysis method of thisembodiment, since liquid L, which is to be analyzed, is dropped onto amirror-finished substrate 14 and the position of liquid L on substrate14 is detected by illumination of light onto substrate 14 and analyzingthe scattered light due to liquid L or trace of liquid L that exists onsubstrate 14, the position of liquid L can be detected even if it isextremely minute in amount. Analysis of an extremely minute amount ofliquid L can thus be carried out smoothly.

[0052] An analysis device 50 of a second embodiment of this inventionshall now be described. FIG. 4 is a diagram, showing analysis device 50of the second embodiment.

[0053] Though analysis device 50 of the second embodiment is the same inbasic arrangement as analysis device 10 of the first embodiment, itdiffers in that second light source 20 is disposed so that light isilluminated from a direction that intersects the normal to the substrateplane. A lens 52 is disposed along the optical axis of the output lightfrom second light source 20 to form an optical system for illuminatinglight onto a measurement spot R on a substrate 14.

[0054] The operations of analysis device 50 of the second embodiment arethe same as the operations of analysis device 10 of the firstembodiment.

[0055] In addition to providing the same effect as analysis device 10 ofthe first embodiment that the position of liquid L that has been droppedonto substrate 14 can be detected smoothly, analysis device 50 of thesecond embodiment provides the following effect.

[0056] That is, with analysis device 50, excitation light is madeincident from a direction that intersects the normal to the substrateplane and the fluorescence that is emitted as a result of thisexcitation light is detected by a fluorescent detector disposed in thedirection of the normal to the substrate plane. Since the reflectedlight of the excitation light will therefore not enter fluorescencedetector 32 directly, the fluorescence emitted from liquid L can bedetected even if it is weak.

[0057] Though embodiments of this invention have been described indetail above, this invention is not limited to the above-describedembodiments.

[0058] Though with the above-described embodiments, a substrate 14,which was coated on the surface with a thin film 14 x formed of PVA, wasused, a silicon substrate 14, 25 having a silicon oxide film (naturaloxidation film) as thin film 14 x, may be used instead.

[0059] With such an arrangement, when a liquid is dropped onto siliconsubstrate 14, a thin film 14 x, comprising silicon oxide, forms at thelocation onto which the liquid was dropped, thus enabling a so-called“watermark” to be left as a trace of liquid L on the silicon substrateand thus even if the liquid evaporates, the trace thereof can bedetected.

[0060]FIG. 5A is a diagram, showing the image of scattered light when asilicon substrate is used, and FIG. 5B is a diagram, showing the imageof fluorescence in the same case. As can be understood from FIGS. 5A and5B, when silicon substrate 14 is used, since the range in whichfluorescence F can be observed will be greater than the range in whichscattered light S can be observed, a range within a fixed radiuscentered about scattered light S must be set as the analysis range. Themagnitude of this radius is determined by the diameter of the liquiddrop formed by the extremely minute amount of the liquid, and, forexample, when the liquid drop diameter is 70 μm, the analysis range ispreferably set to be within a radius of 35 to 40 μm centered aboutscattered light S.

[0061] Also though with the above-described embodiments, substrate 14 ismoved by means of motor-driven movable stage 12 to make measurement spotR be scanned over the entire measurement range of substrate 14, lightmaybe illuminated over the entire measurement range or over a part ofthe measurement range (over a range wider than measurement spot R) ofsubstrate 14 and a plurality of positions onto which liquid has beendropped may be detected in a batch by means of scattered light scatteredby liquid L or traces of liquid L on substrate 14.

[0062] Such an arrangement for batch analysis can be realized byexpanding the illumination range of the output light from each of firstlight source 18 and second light source 20 to cover the entiremeasurement range and likewise expanding the detection range of each oflight detector 30 and fluorescence detector 32 to cover the entiremeasurement range. FIGS. 6A, 6B, 6C, and 6D are diagrams, showing anexample where the measurement range of substrate 14 is analyzed in abatch.

[0063] By illuminating light across the entire measurement range,acquiring the resulting scattered light S by light detector 30 as shownin FIG. 6A, and analyzing this scattered light by means of computer 40,the outlines B of liquid drop traces such as shown in FIG. 6B, aredetermined to detect the positions of liquid L within the measurementrange. By then illuminating the entire measurement range with excitationlight, fluorescence F is acquired in the manner shown in FIG. 6C. Theresults of the analysis using fluorescence F can then be cut out inaccordance with the positional relationship shown in FIG. 6D. By thusperforming batch analysis of substances contained in a liquid, the workefficiency can be improved.

INDUSTRIAL APPLICABILITY

[0064] This invention can be applied to a liquid-contained substanceanalysis device and a liquid-contained substance analysis method.

1. A liquid-contained substance analysis device comprising: amirror-finished substrate, onto which a liquid that is to be analyzed isdropped; a first light source for illuminating said substrate; a lightdetector, detecting scattered light resulting from the scattering oflight from said first light source by said liquid or trace of saidliquid that has been dropped onto said substrate; a position detector,detecting the position on to which said liquid has been dropped based onsaid scattered light detected by said light detector; a second lightsource, illuminating the position detected by said position detectorwith light of a shorter wavelength than the light output from said firstlight source; a fluorescence detector, detecting fluorescence resultingfrom the excitation of said liquid or trace of said liquid by the lightthat has been illuminated from said second light source; and ananalyzer, analyzing substances contained in said liquid by thefluorescence detected by said fluorescence detector.
 2. Theliquid-contained substance analysis device as set forth in claim 1,wherein said second light source is positioned so that the output lightfrom said second light source intersects the normal to the plane of saidsubstrate plane and said fluorescence detector is positioned in thedirection of the normal to said substrate plane.
 3. The liquid-containedsubstance analysis device as set forth in claim 1, wherein the surfaceof said substrate is coated with a water-soluble organic thin film. 4.The liquid-contained substance analysis device as set forth in claim 1,wherein a silicon oxidation film is formed on the surface of thesubstrate.
 5. The liquid-contained substance analysis device as setforth in claim 1, further comprising a stage for setting said substratethereon and moving said substrate so that light from said first lightsource will be illuminated onto said substrate.
 6. A liquid-containedsubstance analysis method comprising:
 7. a liquid dropping step ofdropping a liquid to be analyzed onto a mirror-finished substrate; alight illumination step of illuminating light onto said substrate bymeans of a first light source; a scattered light detection step ofdetecting scattered light resulting from the scattering of light fromsaid first light source by said liquid or trace of said liquid that hasbeen dropped onto the substrate in said liquid dropping step; a positiondetection step of detecting the position onto which said liquid has beendropped based on said scattered light detected in said scattered lightdetection step; a second light illumination step of illuminating theposition detected in said position detection step with light from asecond light source that outputs light of a shorter wavelength than thelight output from said first light source; a fluorescence detection stepof detecting the fluorescence resulting from the excitation of saidliquid or trace of said liquid by the light illuminated in said secondlight illumination step; and an analysis step of analyzing substancescontained in said liquid based on the fluorescence detected in saidfluorescence detection step.